Abstract
BackgroundHuman cystatin C (HCC) is a potential biomarker for tubular damage and impaired renal function. It is difficult to obtain efficient paired monoclonal antibodies against HCC with low molecular to meet the requirements for clinical application The present study was to establish a stable and repeatable measurement for HCC with self-made monoclonal antibodies (McAbs) and Variable domain of heavy chain of heavy-chain antibody (VHHs) increase the sensitivity.MethodsWith hybridoma technology and phage display technology: R-HCC as a screening antigen and N-HCC as the detector for antigens to obtain the specific antibody and established an enzyme-linked immunosorbent assay for human cystatin C using self-made McAbs and VHHs.ResultsWe have successfully obtained three McAbs; 5 F2, 4E4, 1E11 and four VHHs; 3-2, 3-24, 3-33 and 4-5 which were specific for HCC. The measurement of HCC was established with the self-made monoclonal antibodies and VHHs with a high sensitivity the lower limit of detection at 0.5 ng/ml and the detection range at 0.5 ~ 31.3 ng/ml.ConclusionOur data provides a new approach for paired antibody screening and testing of the small molecular biomarker with a single dominant epitope, with the important biological and clinical significance.
Highlights
Human cystatin C (HCC) is a potential biomarker for tubular damage and impaired renal function
Recombinant Human cystatin C (R-HCC) was obtained at a concentration of 2.377 mg/ml and the molecular weight of 17KD, of which the purity was more than 95% (Figure 1C) for Monoclonal antibody (McAb) and VHHs
The steric orthe same decision epitopes of HCC was identified in those McAbs
Summary
Human cystatin C (HCC) is a potential biomarker for tubular damage and impaired renal function. It is difficult to obtain efficient paired monoclonal antibodies against HCC with low molecular to meet the requirements for clinical application The present study was to establish a stable and repeatable measurement for HCC with self-made monoclonal antibodies (McAbs) and Variable domain of heavy chain of heavy-chain antibody (VHHs) increase the sensitivity. Circulating levels of creatinine are considered as one of the common readouts to estimate glomerular filtration rate (GFR), an important evaluation index of renal function [1,2,3,4]. Circulating levels and endogenous clearance of creatinine are used to clinically detect GFR, while there are many factors influencing the accuracy [5,6]. The aim of the present study was to establish a new Double-Antibody-Sandwich Enzyme-Linked immunosorbent assay(DAS-ELISA)-based measurement of HCC with the self-made monoclonal antibody and VHHs by applying the hybridoma technology and phage VHH display technology, to develop a HCC ELISA Test Kit with the highest sensitivity, low cost, and easy operation
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